PROJECT SUMMARY Chronic alcohol abuse is associated with many physiological disturbances, such as sleep disruptions that may aggravate alcohol?s negative effects and potentiate alcohol dependence. Our preliminary results, using a novel chronic alcohol binge-drinking paradigm with concurrent sleep assessments, revealed that sleep/wake fragmentation increases within 2 weeks of alcohol binge exposure in mice. Recently, cortical neural nitric oxide synthase (nNOS) neurons have been identified as key players in the regulation of sleep/wake. Interestingly, nNOS neurons are also expressed in the nucleus accumbens and preliminary results reveal the importance of these neurons in driving sleep homeostasis as well, with nNOS neurons being sleep-active. Interestingly, nNOS is also a molecular target of high interest in the addiction field. Administration of nNOS inhibitors decreases alcohol consumption and blocks alcohol withdrawal, and blocking nNOS expression centrally decreases alcohol intake in rats. nNOS null mutant mice (nNOS-/-) have shown to consume more alcohol than their wild-type counterparts in a free-choice paradigm, while having decreased sensitivity to reinforcing effects of alcohol. Interestingly, chronic alcohol consumption leads to differential effects on nNOS activity across brain regions: nNOS protein levels are reduced in the frontal-parietal cortex, but are elevated within the NAc. Altogether these data lead us hypothesize that alcohol-induced nNOS activity within the NAc may disrupt sleep homeostasis regulation, and thereby mediate alcohol abuse comorbid sleep disturbances. In order to test this hypothesis, we propose to determine whether binge alcohol consumption alters the relationship between sleep/wake state and accumbal vs. cortical nNOS neuronal activity. We will thus assess electroencephalographic (EEG) activity and determine c-FOS expression in nNOS neurons within the Nac and PFC of male and female C57BL/6J mice following acute administration of an intoxicating dose of alcohol, and following 2 weeks of repeated binge drinking. Second, we will assess whether accumbal nNOS neuron ablation decreases alcohol binge drinking and associated sleep fragmentation. We will test the effects of local chemogenetic ablation of nNOS neurons within the NAc on alcohol intake during binge drinking, using micro-injection of a Cre-dependent virus within the NAc of nNOS-CreER mutant mice. Knowledge gained from this exploratory grant will be used as the basis for a larger R01 grant proposal with the ultimate goal of furthering our understanding of neurobiological mechanisms and circuitry of alcohol abuse and comorbid sleep disorders, and to develop novel therapeutics to prevent drug addiction.